Enterococcus faecalis is a leading cause of endocarditis, nosocomial bacteremia and urinary tract infections. Two characteristics contributing to the difficulty in treating enterococcal infections are (1) tolerance to beta- lactam antibiotics and (2) the propensity to cause biofilm-associated infections. The regulation of beta-lactam tolerance and biofilm formation remains incompletely defined. The applicant for this Research Career Award, Dr. Satish K. Pillai, proposes that the E. faecalis quorum-sensing locus fsr, based on its homology to the Staphylococcus aureus regulator agr, also functions as a global regulator. Using the agr paradigm, fsr represents a likely candidate by which E. faecalis regulates pathogenesis-enhancing characteristics like antibiotic tolerance and biofilm formation. Through proposed studies on isogenic fsr-positive and fsr-negative E. faecalis isolates, the applicant will (1) investigate fsr -mediated transcriptional control of biofilm associated genes and evaluate how environmental cues, such as glucose concentration, are relayed through fsr to modulate biofilm formation; (2) characterize fsr regulation of autolysis-dependent and autolysis-independent mechanisms of beta-lactam induced killing; and (3) simulate the conditions of chronic enterococcal infections by developing biofilms in the modified Robbins Device, in order to determine how fsr influences the overall response of E. faecalis to beta-lactam antibiotics, vancomycin, and aminoglycosides. The applicant is an Infectious Diseases fellow at Beth Israel Deaconess Medical Center, and will conduct this research under the mentorship of his sponsors, Drs. Robert C. Moellering, Jr., Roger T. Inouye and Peter F. Weller. In addition to his mentored research, the applicant's career development will by supplemented by course work in microbiology and genetics at Harvard Medical School, bacterial genetics training at Cold Springs Harbor Laboratory, and biostatistics courses at the Harvard School of Public Health. The goals of this application are to (1) define the regulation of enterococcal biofilm formation and beta-lactam tolerance in order to guide strategies to counter this increasingly prevalent pathogen and (2) allow the applicant to develop the skills needed to be an independent investigator.